G De Marco

The sodium-iodide symporter expression in placental tissue at different gestational age: an immunohistochemical study

Background  Iodide (I−) is crucial for foetal thyroid function. Foetal iodide results from maternal circulating iodide and from deiodination of iodothyronines within the placenta. The Na+/I− symporter (NIS) localized in placental cells appears to be involved in iodide exchange. Low NIS expression has been reported in trophoblast cells from the first trimester and pregnancy at term.

TSH receptor and Gsα genetic analysis in children with Down’s syndrome and subclinical hypothyroidism

The prevalence of thyroid diseases in children with Down’s syndrome (DS) is about 3%. The most frequently observed condition is autoimmune subclinical hypothyroidism (SH). Autoimmune SH must be distinguished from defects in the biological activity of the TSH molecule or from the rare inherited condition of thyroid resistance to TSH. To investigate this last aspect we studied 12 patients with DS that had moderately elevated TSH with normal free thyroid hormones without signs of autoimmunity. For the genetic analysis the genomic DNA was extracted from peripheral lymphocytes. All the exons of the TSH receptor (TSHr) and Gsa genes were sequenced. The genetic analysis of the TSHr gene revealed the presence of four polymorphic variants. In two (Pro52Thr — in one patient in the heterozygous state and in the other as a homozygous substitution). In one patient there was an allelic variant in the exon 1 (Asp36His) in the heterozygous state. In 11 patients there was a silent polymorphism in the exon 7 at nucleotide 561. All patients were homozygous for a silent polymorphism in the exon 9 at nucleotide 855. No inactivating mutations of TSHr or Gsa genes were identified in the 12 patients. In conclusion, our results seem to exclude the role of TSHr or Gsa gene mutations in the pathogenesis of the non-autoimmune SH observed in some children with DS.

Ras homolog enriched in striatum inhibits the functional activity of wild type thyrotropin, follicle-stimulating hormone, luteinizing hormone receptors and activating thyrotropin receptor mutations by altering their expression in COS-7 cells

Ras homolog enriched in striatum (Rhes) is a member of the Ras family of small GT-Pases detected in the thyroid. Rhes inhibits signal transduction from Gas protein. In this study we investigated whether Rhes can interfere with stimulation of cAMP/protein kinase A (PKA) pathway of TSH, FSH and LH receptors (TSHr, FSHr, LHr) and of activated TSHr mutants. Receptors were transiently transfected in COS-7 cells with or without Rhes; cAMP was evaluated in basal conditions and after hormone stimulation. Constitutive and bovine TSH (bTSH)-stimulated activity of wild type (wt) and mutated TSHr was inhibited after Rhes co-transfection. Rhes decreased cAMP after FSH and hCG β-subunit (βhCG) stimulation in cells expressing the cognate receptors. In binding experiments Rhes, as another membrane protein, sodium/iodide symporter (NIS), reduced membrane expression of wt TSHr (wtTSHr). In conclusion, Rhes can interfere with the functional activity of wt and mutated TSHr and with the respective hormone-stimulated cAMP production of FSHr and LHr. This interference is not specific and due to the co-expression of two membrane proteins.

Genetic markers to discriminate benign and malignant thyroid nodules with undetermined cytology in an area of borderline iodine deficiency.

BACKGROUND Fine needle aspiration (FNA) with cytologic evaluation is the most reliable tool for malignancy prediction in thyroid nodules, but cytologic diagnosis remains undetermined for 20% of nodules. AIM We investigated the diagnostic potential of a set of 6 marker genes to distinguish benign and malignant thyroid nodules. SUBJECTS AND METHODS The prospective study included 153 thyroid samples obtained by FNA of thyroid nodules from 151 patients (56 benign, 43 malignant, and 54 nodules with undetermined cytology). Gene expression was evaluated by quantitative realtime PCR and statistical analysis of data was performed. All samples were analyzed for V600E BRAF mutation. RESULTS A decrease in TTF3 and HGD1 expression was observed in malignant nodules with respect to benign ones, while an increase in PLAB expression was demonstrated in these nodules. The decision model was valid for 88 of 99 cases of benign and malignant nodules, with a total of 11 false positive or negative predictions. The obtained malignant/benign phenotype prediction was also valid for 37 of 54 cases of nodules with undetermined cytology with a total of 8 false positive and 9 false negative predictions. V600E BRAF gene mutation was demonstrated in 19/43 malignant nodules, in 0/56 benign nodules, and in 1/54 undetermined nodules. CONCLUSIONS The expression profiles of genes (TFF3, HGD1, and PLAB) allowed a good prediction for the differentiation of benign thyroid lesions and thyroid cancer starting from cells of FNA; however, this assay showed limitations when applied to discriminate thyroid nodules with undetermined cytology.

Is resistin a link between highly active antiretroviral therapy and fat redistribution in HIV-infected children?

Objectives: To assess the features of fat redistribution, detected by clinical and ultrasound (US) methods, and the presence of metabolic disorders in HIV-infected children undergoing antiretroviral therapy. To evaluate if serum levels of resistin, a hormone produced only by visceral adipose tissue, are a marker of fat redistribution in these patients. Design and methods: Forty-five consecutive symptomatic HIV-infected children were considered for inclusion in the study. Patients were enrolled if treated for at least 6 months with antiretroviral therapy with or without protease inhibitor (PI) and if compliant to the study protocol. Patients were evaluated for: anthropometric measures, fat redistribution by clinical and US methods, serum lipids, parameters of insulin resistance by homeostasis model assessment for insulin resistance, serum resistin levels by an enzyme-linked immunosorbent assay. Results: Eighteen children fulfilled the inclusion criteria and were enrolled in the study. Twelve (66%) children had clinical and/or US evidence of fat redistribution; 9 (75%) of them were on PI therapy; only 3 of 6 children without fat redistribution were on PI therapy (p<0.05). Serum lipids and insulin resistance parameters did not differ between children with or without fat redistribution. There was a highly significant linear correlation between visceral fat detected by US and circulating resistin levels (r=0.87; p<0.0001). Conclusions: Fat redistribution occurred in most HIV-infected children undergoing PI therapy. Because serum resistin levels reflect the amount of visceral fat, they could be considered a sensitive marker of fat redistribution in HIV-infected children.

Absence of interference of serum IgGs from patients with breast cancer and thyroid autoimmunity on the function of human iodide symporter gene stably transfected in CHO cells

The cause of the association between breast cancer (BC) and thyroid autoimmunity is still unknown. Na+/I− symporter (NIS) is highly expressed in BC cells, and previous studies demonstrated that iodine content in BC is lower than in remote normal breast tissue, suggesting a disorder of iodide uptake in BC. In this study, we evaluated the presence of putative serum autoantibodies able to block the function of NIS in BC patients with thyroid autoimmunity. IgGs were obtained from: a) 11 patients with BC and high antithyroglobulin (TgAb) and antithyroperoxidase (TPOAb) autoantibodies serum concentration; b) 34 patients with Hashimoto’s thyroiditis (HT) (1 was euthyroid, 4 had subclinical hypothyroidism and 29 were overtly hypothyroid); c) 15 control subjects. The biological activity of NIS was studied using a chinese hamster ovary (CHO) cell line stably expressing NIS (NIS-CHO). The course of iodide accumulation in NIS-CHO was studied after addiction of Na125 I in culture medium. The accumulation of iodide linearly increased between 2 and 10 min, reaching a plateau at 45 min. The pre-incubation of NIS-CHO with IgGs purified from sera of BC with the highest levels of TPOAb and TgAb caused an inhibition of iodine uptake of no more than 5%. Similar results were obtained using IgGs purified from patients with HT and control subjects. Our data showed no interference of autoantibodies on iodine uptake in patients with BC and thyroid autoimmunity and the very low percentage of inhibition of iodine uptake cannot explain the lower content of iodine in BC tissue.

Prevalence of activating thyrotropin receptor and Gsα gene mutations in paediatric thyroid toxic adenomas: A multicentric Italian study

Toxic thyroid adenomas or functioning nodules are rare in children and adolescents, representing less than 3% of all causes of hyperthyroidism. Somatic gain-of-function mutations of the TSHr gene (accession number NM_00369) have been identified as a cause of toxic thyroid adenomas and functioning nodules of toxic multinodular goitre in the adult. In patients with toxic thyroid adenomas coming from an area of iodine deficiency, activating somatic mutations in the TSHr gene have been detected in up to 80% and in the Gsa gene up to 25%, suggesting that these genetic anomalies may play a role in the pathogenesis of functioning nodules in adults. In contrast, the incidence of gain-of-function somatic mutations of the TSHr or Gsa genes in rare toxic thyroid adenoma in the paediatric population is poorly understood. In this study, we searched for somatic mutations of TSHr and Gsa genes in a group of nine children affected by toxic thyroid adenomas or functioning nodules that had undergone surgery. Nine children from five Italian paediatric Endocrinology Units who underwent surgery for toxic thyroid adenoma or functioning nodules were included in this study. At diagnosis, six patients were clinically thyrotoxic and three had normal FT4 and FT3 and low TSH serum levels. Thyroid glands were studied by clinical examination, thyroid ultrasound, scintiscan imaging using iodine-123 (I) or technetium-99 m (Tc) and histology. Thyroid autoimmunity was excluded by the absence of circulating antithyroid antibodies. Genomic DNA was extracted from formalin-fixed paraffin-embedded thyroid sections, and direct sequencing of exons 9 and 10 of the TSHr gene and exons 8 and 9 of the Gsa gene was performed. The TSHr gene mutant harbouring the single-nucleotide missense substitution was generated by site-directed mutagenesis; COS-7 cells were transiently transfected using the DEAE-Dextran technique and used for cAMP production assay, I-bTSH binding and microchip flow cytometry analysis. Two heterozygous TSHr gene point mutations were identified in genomic DNA from nodular tissues with a prevalence of two of 9 (about 22%). No alteration was identified in normal tissues or blood cells. A residue change at position 568 of the TSHr (I568F) due to the heterozygous nucleotide substitution ATC/ TTC (Fig. 1a) was revealed in one nodule. In genomic DNA from another nodule, a residue change at position 453 of the TSHr (M453T) due to the heterozygous nucleotide substitution ATG/ACG (Fig. 1a) was identified. Sequence of exons 8 and 9 of the Gsa gene was normal in each patient. The I568F mutant was functionally characterized in this study, while the functional characteristics of M453T mutation were well known. The I568F mutant was characterized by increased basal constitutive activity in terms of cAMP accumulation when compared with the

Patients affected by vitiligo and autoimmune diseases do not show antibodies interfering with the activity of the melanocortin 1 receptor

Background: Vitiligo is an acquired depigmenting disorder characterized by the loss of melanocytes from the epidermis with the development of white patches in various distribution. The pathogenesis of vitiligo is still unknown, but the association with autoimmune disorders and organ specific autoantibodies, supports the hypothesis of an autoimmune pathogenesis. Aim: The aim of the present study was to investigate if autoantibodies present in sera of patients affected by vitiligo may be able to interfere with the activity of the αMSH on the melanocortin 1 receptor (MC1R). Materials/Subjects and methods: IgG from the sera of 41 patients with vitiligo associated or not with thyroid autoimmune diseases or other autoimmune pathologies were incubated with HBL20 cells (human malignant melanocytes expressing the MC1R) in the presence of a sub-maximal dose of αMSH. A normal IgG range was determined by using IgG extracted from 30 control sera of normal subjects. Results: None of the IgG from vitiligo patients was able to inhibit αMSH-stimulated cAMP production in HBL20 cells. Conclusions: Autoantibodies against MC1R are rare or absent in sera of vitiligo patients.

Sequencing of the entire coding region of the receptor associated protein (RAP) in patients with primary hypothyroidism of unknown origin

The LDL receptor-associated protein (RAP) is involved in secretion of thyroglobulin (Tg) from the thyrocyte to the colloid. Disruption of the RAP gene in mice results in a reduced Tg content within the colloid, leading to subclinical hypothyroidism and histological alterations resembling early goiter. Here we studied the entire coding sequence of RAP in genomic DNA samples from 18 patients with primary hypothyroidism not due to thyroid autoimmunity or dysgenesis. the control group included 21 subjects with no evidence of thyroid alterations. Eleven different polymorphisms with amino-acid substitution and 4 different missense polymorphisms without amino-acid substitution were found in various regions of the RAP gene. Only one polymorphism in exone 7 (V311M) was observed exclusively in patients, but it had been previously reported in normal subjects as well. The remaining polymorphisms were found either both in patients and controls or only in controls and had not been previously reported. The frequency of the various polymorphisms did not differ significantly between patients and controls. based on these findings, we conclude that alterations of the RAP gene are not a common cause of hypothyroidism, although it cannot be excluded that other, rarer alterations with a pathogenic effect exist, and that they should be investigated in a larger number of patients.

STUDY OF POTENTIAL INHIBITORS OF THYROID IODIDE UPTAKE BY USING CHO CELLS STABLY EXPRESSING THE HUMAN SODIUM/IODIDE SYMPORTER (hNIS) PROTEIN

Background: Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to “endocrine disruptor” action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. Aim: The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. Materials and methods: A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. Results: None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. Conclusions: In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NIS-mediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner.